Purification and characterization of honey bee vitellogenin

Abstract

A protocol has been developed for the purification of vitellogenin from the honey bee, Apis mellifera. Purification allows for the first characterization of a vitellogenin from the large order Hymenoptera. Hymenopteran vitellogenins are unusual among insect vitellogenins in that they contain only one type of apoprotein. The honey bee vitellogenin was isolated from hemolymph of honey bee queens by a combination of density gradient ultracentrifugation, ion-exchange chromatography, and affinity chromatography. The native vitellogenin particle is a very high density glycolipoprotein containing approximately 91% protein, 7% lipid, and 2% carbohydrate. Phospholipid and diacylglycerol are the major lipid components. The equilibrium density (1.28 g/ml) is the same as that for Manduca sexta vitellogenin, which contains a much higher proportion of lipid. The covalently bound carbohydrate moiety of the particle is high in mannose. The amino acid composition of vitellogenin is similar to those of vitellogenins from other insect species. The N-terminal amino acid sequence of the apoprotein was determined, the first such sequence for any insect vitellogenin. When analyzed by sodium dodecyl sulfate (SDS)-gel electrophoresis, A. mellifera vitellogenin resolved into a single band with an apparent Mr of 180,000. Gel filtration under reducing and native conditions yielded estimated Mr values of about 300,000.

A vitellogenin polyserine cleavage site: highly disordered conformation protected from proteolysis by phosphorylation

“…Cleavage in the vicinity of the polyserine region occurs naturally in the bee fatbody tissue and during AmVg purification (Havukainen et al, 2011;Wheeler and Kawooya, 1990). In contrast to the Vg expressed by many other species, AmVg does not have the consensus cleavage site (Barr, 1991;Rouille et al, 1995).…”

Downregulation of vitellogenin gene activity increases the gustatory responsiveness of honey bee workers (Apis mellifera)

“…Hemolymph was dissolved in 50 μl TRIS-buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 5 mM benzamidin, 0.7 μM pepstatin, 8 μM chymostatin, 10 μM leupeptin, and 0.8 μM aprotinin, Sigma-Aldrich), before samples were separated by 7.5% SDS-polyacrylamide gel electrophoresis using standard methods [26]. A β-galactosidase standard (Sigma-Aldrich) was included to allow densitometrically quantification by the method of Lin et al [27], in which vitellogenin is detected as a single band of 180 kDa [27,28]. The densitometrical analysis was performed by the Quantity One imaging software (Bio-Rad) after staining the gels with Commassie Brilliant Blue (Sigma-Aldrich).…”

Deconstructing honeybee vitellogenin: novel 40 kDa fragment assigned to its N terminus

“…In terms of biochemistry, honeybee vitellogenin is grossly described as a 180kDa monomeric phospholipoglycoprotein (Wheeler and Kawooya, 1990). All insect vitellogenins, excluding those of the honeybee suborder Apocrita, are cleaved in vivo, typically close to the polyserine track(s) at an RXXR consensussequence motif by subtilisin-like endoproteases (Barr, 1991;Rouille et al, 1995).…”